The present invention relates to the genical fields of molecular imaging and genetic sequencing.
Current methods of nucleic acid sequencing and mapping, and protein and tissue imgaging, are based on radioactive, bio- and chemiluminescent emitters, photographic plates, and some electronic techniques. None of these have in practice been found to be entirely satisfactory.
Fluorescent imaging, radio labelling and bio- and chemiluminescent markers used with film and/or emulsion are expensive, very slow, limited and difficult to interface to computers. The techniques involved are difficult, and require hazardous handling and disposal procedures requiring substantial technical expertise. The materials used are often difficult and expensive to obtain, and have short shelf lives. Photographic imaging, which is frequently used, takes days or sometimes weeks or months to accomplish, and is limited by virtue of its small dynamic range and relatively poor linearity of response.
Of the electronic techniques, phosphor imaging/multiwire proportional chamber (MWPC) and the microchannelplate/MWPC approaches are unpopular with many molecular biologists because of their limited linearity and cost.
It may be helpful by way of background to set out in some detail the current state of the art in nucleic acid imaging. There are two separate methodologies which are currently in common use, details of which are set out below.
First, there are those involving the use of a photographic film to visualise chemiluminescent or radioactive labels and secondly, those using a light emitter, such as ethidium bromide, which chemically binds to nucleic acids and emits orange light under UV stimulation. The first imaging technology is usually used in nucleic acid sequencing. This process involves the chemical labelling of a component of the sequencing reaction, usually the primer, with a radio-isotope or chemiluminescent marker, or tag. Subsequent to the sequencing reaction and electrophoresis, this tag is used to image the position of the nucleic acid bands by the exposure of normal photographic film to the dried electrophoresis gel. This is a lengthy process, taking from 24 to 72 hours, depending on the sequencing technique used. Many of the radioactive markers used in nucleic acid sequencing are extremely hazardous and introduce extra complications to the sequencing process therefore any imaging system which removes the necessity for these markers would be extremely advantageous.
The second technology, that of light emitters, is generally used for DNA restriction analysis and plasmid construction planning. This technique relies on the imaging of nucleic acids in simple agarose gels using the carcinogenic chemical ethidium bromide which emits orange light after UV stimulation to visualise the size and estimate the concentration of nucleic acid present. Ethidium bromide is a highly undesirable component of this technique with dangerous accumulative medical consequences. Its removal from the imaging technique would be very advantageous in every application of agarose gel analysis.
Nucleic acid sequencing, as opposed to spatial imaging, normally makes use of a rather different process. This process involves the chemical labelling of a component of the sequencing reaction, usually the primer, with a radioisotope or bio or chemiluminescent marker, or tag. Subsequent to the sequencing reaction and electrophoresis, this tag is used to image the position of the nucleic acid bands by exposing photographic film to the dried electrophoresis gel. This is a lengthy process, taking from 24 to 72 hours depending on the sequencing technique used.
DNA restriction enzyme analysis (DNA mapping) and vector construction is a fundamental aspect of molecular biology. These mapping techniques also rely on the photographic imaging of nucleic acids fragments in simple agarose gels using ethidium bromide. This marker emits orange light after UV stimulation, to visualise size and estimate concentration of nucleic acids. Ethidium bromide is an undesirable component of this technique with dangerous cumulative carcinogenic consequences. Its removal from the imaging technique would be very advantageous in every such application of agarose gel analysis.
The imaging of peptides and proteins is often the end destination of many genetic engineering processes. However, it also forms a huge portion of general biological, biochemical and medical research. Indeed, it is hard to think of a bioscience area that is not dependent, at least in some way, on protein analysis. The analysis is again normally based on electrophoretic techniques, being dependent on the addition of a marker. Generally these are radioactive although chemiluminescence and specialised chemical stains are also used.
The field of tissue imaging is hugely important in the areas of drug development, and medical and molecular diagnostics. Traditionally, xc3xa2-emitting markers may be used to image the distribution of a drug within a tissue sample. This process typically takes weeks or even months, and requires the use of potentially hazardous substances.
It will be understood that all of the present methods mentioned above require the use of either radioisotopes or other hazardous substances in addition, at least some of the techniques listed require the use of expensive and inconvenient electrophoresis gels. To summarise, the major problems are as follows:
1. Training. Health and Safety standards require all workers in contact with any form of radioactivity to have extensive training in the handling, use and disposal of radioactivity.
2. Use. The incorporation of radioactivity into a system is often a complex and time-consuming process. The worker must take extreme precautions, for example with the isotope 32P, which is commonly used in DNA sequencing, has to be shielded from the worker by perspex, which makes an already complex experiment much harder. Subsequent to the initial experiment, the further manipulation of the already fragile electrophoresis gel is complicated by the radioactive present. Radioactivity can be replaced by chemi- or bio- luminescent imagers, but these, and while these are safer they are still complicated to use.
3. Time. All these imaging systems rely on the use of autoradiography to visual the nucleic acids or proteins. This is achieved by drying the gel onto a piece of filter paper and exposing it to a piece of photographic film. The film must be exposed for anything between 24 hrs to 3 months. Therefore it can take from days to months to even see if the experiment worked. This is a major problem in molecular biology and increases the length of research projects significantly.
4. Expense. The various components of these experiments are expensive. Radioactivity has a limited shelf-life because of its natural decay, and it is also expensive, with 35S costing about L250 for 20 sequencing reactions. The film used is also very expensive as some of it measures 35xc3x9745 cm.
5. Disposal. These processes generate large volumes of solid and liquid waste, all of which must be disposed of legally and responsibly. This is also very expensive and troublesome.
According to a first aspect of the present invention there is provided a method of imaging molecules within a biological sample comprising shining a UV light onto the sample, and detecting the position of molecules of a selected class by the molecular UV absorption of molecules of that class.
According to a second aspect of the present invention there is provided a molecular imaging device for imaging molecules with a biological sample, comprising a UV light source arranged to shine onto a sample to be investigated and a UV detector arranged to detect the position of molecules of a selected class by the molecular UV absorption of molecules of that class.
According to yet a further aspect of the present invention there is provided an electrophoresis apparatus comprising an electrophoresis material onto which samples to be analysed are loaded, means for applying a potential difference along the material thereby causing the samples to move (migrate or drift) along the material, and a fixed detector located part way along the material and arranged to detect molecules of a selected class as they move past the detector.
According to yet a further aspect there is provided an electrophoresis apparatus comprising an electrophoresis material onto which samples to be analysed are loaded, means for applying a potential difference along the material thereby causing the samples to move (migrate or drift) along the material, a light source arranged to shine onto the material and a detector arranged to detect the position of molecules of a selected class by the molecular light absorption of molecules of that class.
In the preferred method of the present invention, molecules are imaged by detecting their intrinsic absorption of UV light. In this aspect of the invention, we use the intrinsic image of the molecule itself, whether it be a nucleic acid fragment, a protein, or indeed a polypeptide chain. The image comes from the absorption of that molecule, using molecular UV absorption spectrometry, in contrast to the well known technique of optical spectromety. The key advantage is the lack of a tag.
This has many important consequences. Perhaps the most obvious is that no hazardous tag is no longer needed, whether it be radioactive or a well known carcinogen like ethidium bromide. Another issue is that, for sequencing reactions, the lack of the tag removes one of the major constraints on the number of bases that can be sequenced; that is, the amount of radioactivity that can be incorporated within the sample.
Preferably, the molecules of interest are directly imaged by detecting their absorption by imaging the nucleic acid, protein or tissue map onto a diamond detector. This may be accomplished by illuminating the object to be imagedxe2x80x94whether it be nucleic acids, proteins or tissue, with constant brightness UV light from either a broad spectrum device like Helium discharge tube, or a monochromatic laser like an excimer laser at 196 nm. We observe the different amounts of light reaching a detector placed behind the object being imaged. In the case of two or three dimensional imaging, the shadow is imaged simultaneously, and the object thereby identified. This requires a two-dimensional detector, like a pixel device or a pixellated ridge device.
In the superior case of the directional laser, we can scan the object to be identified onto a one dimensional detector, either planar (with strip electrodes) or ridged, and build up a two dimensional image. The latter case is superior in an additional way: by making two (or more) scans with non-parallel laser beams, a stereoscopic image can be made, allowing 3D reconstruction.
When the invention is applied to nucleic acid manipulation and quantitation, the technology will allow a quantitative differentiation between transmitted and absorbed energy. The quantitation of DNA under these conditions has important applications in the construction of expression vectors that are used to produce specialised proteins used in therapeutics.
When the present invention is applied to the imaging of peptides and proteins, the same equipment may be used as that used to image DNA restriction enzyme maps. The speed of imaging provided by the present invention offers significant advantages over existing techniques, some of which can take up to three months.
This idea is based on the absorption by proteins of UV light at  less than 230 nm, also an optimal range for nucleic acids. Thus the same detector could image both types of molecules. An added bonus of the feature is that lipids, carbohydrates and other small macromolecules absorb UV poorly if at all in this range, thus allowing an intrinsic filtering of biological noise on the image.
This spectral response is ideally matched to that of diamond, which turns on at 224 nm, and is extremely insensitive to light of greater wavelengths or lower energy. This is a very powerful attribute for a detector to have. A typical wavelength range for a diamond detector is about 180-224 nm, but since the lower limit is imposed more by the materials and/or source than the detector medium, lower wavelength detection may not be excluded in all circumstances. For certain applications, silicon detectors could be used (detection range 190-300 nm). Photomultipliers might also be used.
In its application to tissue imaging, two-dimensional images may be built up using a pixelated detector, or a strip in which the source is scanned orthogonally to the strips. In the preferred embodiment of the invention, a diamond detector is used, and two-dimensional images are built up by pixellating the diamond or scanning the UV source orthognally to the strips.
Mostly usefully, three dimensional stereoscopic images can be built up by the addition of images from non-parallel lasers imaged on the same detector. In this case, we propose to strobe alternately the lasers, with the detector read out with the parallax shifts determining the three-dimensional structure. A highly resolved three-dimensional image will allow details on the tissue structure to be derived. This will greatly cut down development time for a new drug and hence reduce the overall cost.
In one preferred embodiment, the invention extends to an electrophoresis apparatus, for example to a DNA sequencer. The apparatus preferably monitors qualitatively the changes in a signal from a source as bands of nucleic acids passed between the source and the detector on an electrophoresis gel. As the bands pass the detector, they may be digitised directly to a data base.
The apparatus may further incorporate the concept of using inert resusable solid phase for the electrophoresis. For a DNA sequencer, the solid phase may be coated or otherwise supported on a quartz substrate (for example a tube), there preferably being four separate tubes for the four bases.
The present invention, in one or more of its preferred forms, has the ability to image Nucleic Acids (including DNA restriction enzyme fragments, Polymerase Chain Reaction (PCR) products, DNA sequence reaction products, any other single or double-stranded DNA polymer molecules and messenger RNA transcripts). The invention also provides the possibility for imaging proteins (including protein digest fragments, expressed proteins, expressed fusion proteins, peptides, amino acids and cell extracts). In both cases, electrophoretic solid phases are preferably used, and the imaging is carried out by the intrinsic UV absorption of the Nucleic Acid or the Protein, respectively. The invention also provides the ability to quantify the amount of Nucleic Acid and/or Protein.
The electrophoretic solid phase may be held in a capillary, a microchannel chip structure, between plates in a standard DNA/RNA gel or in a one dimensional or a two dimensional protein gel. The invention is also applicable for use with other gel systems.
The solid phase may be agarose, polyacrylamide or any other suitable solid phase matrix.
A visualisation system has been developed which in one embodiment acquires real time images of unstained and unlabelled peptide and protein fragments, protein bands and 2-Dimensional (2D) cellular proteomic maps in electrophoretic gels. As well as visualising the location of these biomolecules, the system also allows their quantitative analysis through a built-in software system. The accuracy and sensitivity of the system, in its preferred embodiments, allows the detection and quantification of proteins that are symptomatic of diseased states within the individual from which the proteins came. In addition, the system is capable of measuring and quantifying the effect on proteins of nutrients, environmental factors and drugs. As applied to disease diagnosis, the system may, amongst other things, be applied to proteins that have been extracted from human blood serum, and the protein changes measured which may symptomatic of various melanomas including breast cancer. This provides the possibility of early detection without the need for intrusive biopsies.
This system can be implemented statically, in which a single image is taken or dynamically, in which a series of timed images are acquired revealing information on the individual proteins spots"" mobility (indicating modifications) as well as their presence or absence. By making us of digital camera technology (e.g. using CCD arrays), imaging within a few micrometer accuracy is possible, as high quantum efficiency. The system is designed to replace existing technologies which rely on labelling the protein molecules with an extra component such as a radioactive or fluorescent markers, or by post-electrophoretic staining of the polyacrylamide gels by chemical dyes such as Coomassie Blue. These techniques are handicapped by a series of drawbacks which are overcome by the Intrinsic Imaging approach which allows the user to see the actual protein, not the label or the stain.
To summarize, the following are the primary advantages of the present invention, or subsidiary aspects of the present invention:
1. Training. The removal of radioactivity from the systems obviates the need for Health and Safety training.
2. Use. The removal of radioactivity or any other extrinsic imaging component from the experimental process dramatically increases the efficiency and speed of those reactions. The labelling step (where the radioactive marker is added to the reaction) is often complicated, and its failure cannot be perceived until the end of the experiment.
3. Time. The ability to image the results of, for example, a sequencing gel in minutes rather than hours is expected to lead to a dramatic increase in the efficiency of large scale sequencing operations such as the Human Genome Project. It would also allow the faster discovery of problems within the reractionxe2x80x94a lot of time is lost in molecular biology due to the time it can take to realise that an experiment has not gone according to plan.
4. Expense. The application of hardware based on this technology would lead to a massive release of funds from any research groups consumables budget. Following the initial equipment costs, many molecular biology groups could expect to see their radioactivity and film requirements drop substantially.
5. Disposal. Environmentally, the benefits of the technology are immense. The total removal of radioactivity from the system of course eliminates the need for its disposal.
6. Maintenance of biological activity. Another major advantage of the technique is the detection of nucleic acids and proteins in a system where their biological activity is not comprises by the presence of a label. Therefore biomolecules can be detected, imaged and recovered for analysis without undergoing any biochemical modifications that might destablise or affect their functionality thus devaluing their utility in downstream analysis. This allows the user to recover meaningful data from electrophoretic gels and obtain functional proteins at the same time, giving a huge saving in time and resources.
7. Protein Elution and Purification. Recovery of individual proteins from an SDS-PAGE gel is a complex technique where some of the sample is inevitably lost. The lost protein is then compounded by further attrition as the binding of stain or label results in a biochemical alteration of the state of the protein. The use of Label Free Intrinsic Imaging results in a higher recovery rate of an unadulterated individual protein from, for example, a 2D proteomic gel.
8. Real time Electrophoretic Monitoring. Existing systems cannot easily monitor the migration of biomolecules in situ in an electrophoretic gel because they are not inherently digital and they are incapable of seeing the actual protein only the label. If a labelling technique has been employed, the gel must be imaged post-electrophoresis, by for example, autoradiography (for a radioactive label). This involves stopping the electrophoretic process which cannot then be restarted after the proteins have been imaged. If the gel has not been run for long enough, the samples can be lost. Our approach, because it visualised the proteins in the electrophoretic gel as they migrate, allows the exact extent of migration to be defined by the user.
9. Quantification. The ratio of signal to quantity is not easily definable when a labelling technique is used. This is because the molar ratio of label to protein molecule may not be constant. Our approach allows a direct and unambiguous quantification of the exact amount of protein present in the gel as it migrates.
10. Degrees of freedom. Healthcare monitoring relies on the processing of large numbers of gels by low-grade technicians. The correct and reproducible staining and imaging of gels in, for example a cancer screening system, is dependent upon this human factor. Mistakes or carelessness in the staining process can result in misinterpreted analysis. Removing the staining process eliminate one complete source of error, which can have tragic results if false negatives allow a disease state to progress past the point of return. The inherent digitisation of the preferred system also allows the automation of large parts of this process. Pattern recognition software may be used to allow the removal of the human factor from large parts of the disease monitoring process.